Amyloid fibrils are an insoluble, highly ordered, fibrous protein structure, which have increasingly been recognised as having bionanotechnology applications. Their ability to selfassemble allows a bottom-up approach to material design. Their nanometre dimensions affords them a high surface-to-volume ratio and their proteinaceous building blocks from which they are assembled allow for decoration with biomolecules and chemicals through amino acid residues. Amyloid fibrils are therefore a potential nanoscaffold for immobilisation of biomolecules. Immobilisation offers a solution to the problems associated with the use of enzymes in in vitro applications, by increasing their stability, reusability, and in some cases, enhancing catalytic activity. Nanosupports offer a high surface-to-volume ratio compared to classical planar 2-D supports, potentially affording them dramatic increases in immobilisation capacity. To investigate the potential of amyloid fibrils as a novel nanoscaffold, organophosphate hydrolase (OPH), cytochrome P450BM3 (P450BM3), green fluorescent protein (GFP), tobacco etch virus protease (TEV), and glucose oxidase (GOD) were immobilised in solution to the model amyloid fibril forming protein, bovine insulin. Covalently immobilised OPH was found to have a ~300 % increase in relative thermostability at 40 and 50 °C. P450BM3 was not successfully immobilised in its active state, most likely due to unfolding of the enzyme on the amyloid fibril surface. Covalently immobilised GFP retained full fluorescence and acted as a fluorescent protein tag. TEV was shown to have a physical interaction with the nanoscaffold and retain activity. GOD was immobilised and retained activity. Although not all proteins retained activity, a range of different protein structures were successfully immobilised onto the insulin amyloid fibril nanoscaffold. Attachment to the crystallin amyloid fibril nanoscaffold remains a work in progress due to the complexities associated with post-translational modifications of these fibrils. Crystallin amyloid fibrils were assembled on a surface for the first time. Their surface assembled structure was found to resemble spherulites, not previously seen before with crystallin amyloid fibrils. Bovine insulin amyloid fibrils were assembled on the surface of glass beads to increase the available surface area for biomolecule immobilisation. The surface assembled bovine insulin nanoscaffold was first functionalised with GOD, demonstrating that the nanoscaffold provides more surface area for biomolecule immobilisation, although in this case the increase was limited due to high non-specific binding of GOD to the unmodified glass surface. GFP was successfully employed as a fluorescent protein tag to assess the degree of nanoscaffold coverage, confirming the nanoscaffold affords the glass bead a greater surface area. Moreover, a reusable immobilised TEV protease-bead system was developed that was able to sequentially cleave the poly-histidine tags of three different proteins. In conclusion, bovine insulin amyloid fibrils have been shown to be a versatile nanoscaffold for the immobilisation of a range of biomolecules. The surface characteristics of the nanoscaffold allows for both covalent and physical immobilisation of biomolecules. Thus, amyloid fibrils have exciting potential in the creation of novel bionanotechnologies.

L'objectif de cette thèse est de synthétiser de nouvelles surfaces spécifiques nécessaires à l'immobilisation des biomolécules ; visant à développer à terme un biocapteur pour la détection de pathogènes en industrie agroalimentaire. Cette nouvelle procédure de fonctionnalisation de surface consiste d'une part à greffer des molécules organiques sur un substrat métallique à partir d'une réaction électrochimique et d'autre part de synthétiser un monomère photopolymérisable sur tout type de surface. Ces surfaces sont enfin utilisées pour immobiliser les biomolécules. Ce procédé ainsi développé permet d'éliminer les multiples étapes, l'utilisation excessive de réactifs observés dans les protocoles classiques de fonctionnalisation de surface pour la capture de microorganismes. Deux stratégies de fonctionnalisation ont été investiguées : la polymérisation sur une plaque de platine et le dépôt de monocouche sur une surface d'or. La fonctionnalisation de surfaces ainsi que l'immobilisation de biomolécules ont été caractérisées par la spectroscopie Raman, la microbalance à cristal de quartz, la microscopie à force atomique (AFM) pour le premier et en plus la microscopie à fluorescence pour le second. Les résultats de la fonctionnalisation de surfaces par dépôt de polymère ont montré, une déstabilisation du polymère en présence de l'eau. Afin d'optimiser la synthèse, nous avons travaillé en milieu inerte, sous alumine activée. De plus, on note une large couverture de la zone spectrale des biomolécules par les signaux du polymère ; Pour le dépôt de monocouche, l'on a obtenu une surface très réactive, homogène. La diffusion Raman est la principale technique de caractérisation utilisée. Elle présente l'avantage d'être une méthode de caractérisation physico-chimique non destructive et non invasive. Longtemps délaissée dans les sciences du vivant, cette méthode apparaît maintenant particulièrement prometteuse grâce à un développement récent de spectromètres intégrés performants. La diffusion Raman sur la monocouche déposée montre une intensité accrue des signaux par l'utilisation de la surface d'or et un spectre plus dégagé conduisant à l'identification aisée des biomolécules après fixation. Elle permet non seulement d'identifier les bandes de vibrations de chaque groupement mais aussi la conformation des structures. Les résultats d'immobilisation ont montré que l'accroche des biomolécules sur les surfaces fonctionnalisées était spécifique. La fonctionnalisation de surface d'or par dépôt de monocouche constitue finalement une technique très rapide à mettre en œuvre, peu coûteuse permettant d'ancrer efficacement les biomolécules et peut être utilisée pour diverses applications. La synthèse du monomère photopolymérisable a été abordée et est en cours d'investigation.

Riparian buffer zones, adjacent to waterways, may protect water quality by intercepting and removing nitrogen in runoff from agricultural land. This research comprised four parts: (1) a field study of nitrogen buffering by differently vegetated riparian zones in a United Kingdom (UK) sheep-grazed pastoral catchment; (2) a field study of surface and subsurface runoff hydrology, and nitrogen flux, in a UK riparian wetland; (3) a laboratory study ((^15)N tracer-isotope dilution) of microbial inorganic nitrogen production and removal processes in the UK riparian wetland soil; and (4) a laboratory microcosm study ((^15)tracer) of nitrate removal processes in bare and plant-inhabited (Glyceria declinata) New Zealand (NZ) riparian wetland soil. Dissolved organic nitrogen and ammonium were generally more important components of subsurface runoff than nitrate in the three UK riparian zones. All riparian zones were poor buffers having minimal effect on the nitrogen concentration of subsurface runoff. In the UK riparian wetland site subsurface (saturated zone) and surface 'preferential flow paths' typically conveyed large quantities of catchment runoff rapidly into, and across the site, and hindered nitrogen buffering. However, under low flow conditions, runoff-riparian soil contact increased and the wetland decreased the catchment nitrogen flux by 27%. In the UK riparian wetland soil most nitrate removal was attributable to denitrification (87- 100%) as opposed to dissimilatory nitrate reduction to ammonium (DNRA) (0-13%) and immobilisation (0-10%). Total ((^14)N+(^15)N) transformation rates for these processes were 1.3-47, 0.5-1.5 and 0.6-2.5 μg N g soil(^-1) hr(^-1) respectively. In the NZ riparian wetland soil Glyceria declinata assimilated 11-15% of nitrate but, more importantly, increased soil oxidation and altered the proportions of nitrate removal attributable to denitrification (from 29% to 61-63%) and DNRA (from 49 to <1%), but not immobilisation (22-26%). Denitrification and, thus, nitrogen buffering might be enhanced, in some riparian zones by increasing the extent of moderately anoxic soil with plants that release oxygen from their roots or with water table management.

The onset of the Water Framework Directive (WFD) will challenge water utilities to further reduce their wastewater phosphorus discharges to < 0.5 mg.L- 1. Whilst conventional treatments, such as chemical dosing, are able to meet these new discharge consents, the strategies are representative of a linear economy model where resources are unrecovered and disposed. An alternative solution which can contribute to the aspiration of a circular economy is microalgae. Microalgae are ubiquitous in wastewater environments and assimilate phosphorus during their growth, to residual concentrations complementary of the WFD. Furthermore, microalgal biomass can be anaerobically digested to produce biomethane offering the potential for an energy neutral approach. However, uptake of microalgal systems are lacking in the UK through limited knowledge of operation; and the belief that such solutions are synonymous to large, shallow open ponds with extensive treatment times. The development of alternative microalgal reactors are increasingly investigated to overcome these implementation challenges. Of these, immobilised microalgae has shown great potential; and whilst within its infancy demonstrates the greatest opportunity for development and optimisation. This thesis determines the critical operational parameters that influence the remediation efficacy of immobilised microalgae for tertiary nutrient removal; including species selection, biomass concentration, treatment period and lighting; with recommendations for optimal performance. These recommendations are then applied to the design and operation of an immobilised bioreactor (IBR) to understand the key design and operating components that influence the overall economic viability. In doing so, the potential for an IBR to be economically viable, within the next decade, in comparison to traditional approaches are discussed.

No / Purpose: The immobilisation and restraint of children to facilitate radiographic examination is a controversial issue that has been relatively ignored by radiography research. The aim of this study was to begin to fill this gap by providing a description of restraint used in a limited number of clinical sites in order to highlight any perceived need for training, policies or guidelines in the use of child immobilisation and restraint. Methods: A cross-sectional survey design using a postal questionnaire was adopted. One hundred and sixty-seven questionnaires were distributed to radiographers employed within six hospital Trusts. Results: A response rate of 83.2% (n=139/167) was achieved. Ninety-three percent (93.5%, n=130/139) of respondents indicated that restraining techniques were used although only 19.2% (n=25/130) had received specific training in safe restraining techniques and 7.9% (n=11/139) in distraction techniques as an alternative to restraint. A need for further guidance and support for clinical staff was evident with 73.3% (n=74/101) of respondents identifying a need for specific guidelines and 84.6% (n=110/130) indicating that further training opportunities were required. Conclusions: The use of restraint in paediatric plain film radiography is an apparently widespread practice and support for clinical radiographers through the development of training opportunities and practice guidelines are seen as essential in order to promote high quality paediatric radiography practices.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: ß-Galactosidase of Escherichia coli is the equivalent of lactase in humans and has the ability to bind and hydrolyse lactose. Lactase de ciency is a common phenomenon present in almost 70% of the world's population. This has resulted in greater than before demands on the food processing industry to develop a method that will allow for the hydrolysis of the disaccharide lactose in milk but will also allow for the removal of the remaining active enzyme. In this thesis, a new method, that is bio-speci c and well characterized for the removal of lactose from a lactose containing solution, is described. The E537D mutated version of ß-Galactosidase, which has a much lower activity compared to the wildtype and is able to bio-speci cally bind lactose for longer periods, was covalently immobilised to commercially available magnetic nanoparticles (fl uidMAG-Amine) via two coupling strategies. Glutaraldehyde is a cross-linking agent that reacts with amine groups, while N- (3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is a coupling agent that activates carboxylic groups. These agents are widely used for the coupling of biomolecules to solid supports. The covalently coupled fluidMAG-E537D ß-Galactosidase particles were characterized regarding retained enzymatic activity and ability to bind and physically remove lactose from a lactose containing solution by applying an external magnetic eld, after lactose binding, to the enzyme-particle complex in solution. Each component aimed at yielding this functionally immobilised enzyme complex was studied and optimized to contribute to the development of this novel technique, which is a ordable and simple, for the removal of lactose from solution for the ultimate production of lactose free milk. Results indicated the glutaraldehyde method of ß-Gal cross-linking to fluidMAG-Amine to be the preferred strategy since it allowed an increased carrier capacity of protein to the particles. The glutaraldehyde cross-linked protein also exhibited a two-fold higher activity than the EDC coupled protein. Furthermore, the glutaraldehyde cross-linked fluidMAG-E537D ß-Gal was able to physically remove 34 % of the lactose from a 0.2 nmol/L lactose in solution. This, therefore, con rmed the potential use of this novel technique in the food processing industry. / AFRIKAANSE OPSOMMING: ß-Galaktosidase vanaf Escherichia coli is dieselfde as laktase in mense en beskik oor die vermoë om laktose te bind en te hidroliseer. 'n Gebrek aan laktase kom algemeen voor en ongeveer 70 % van die wêreldbevolking ly hieraan. Laasgenoemde het daartoe gelei dat daar meer druk as vantevore op die voedselproduksie industrie is om 'n metode te ontwikkel waarmee die hidrolise van die disakkaried laktose in melk moontlik sal wees asook die verwydering van die oorblywende aktiewe ensiem. In hierdie tesis word 'n nuwe metode beskryf wat biospesi ek en goed gekarakteriseer is vir die verwydering van laktose vanuit 'n laktose bevattende oplossing. Die E537D gemuteerde weergawe van ß-Galaktosidase, wat beskik oor 'n baie laer aktiwiteit as die wildetipe asook die vermoë om laktose biospesi ek vir langer periodes te bind, is kovalent geïmmobiliseer op kommersieel beskikbare magnetiese nanopartikels (fluidMAG-Amine) via twee koppelingsstrategieë. Glutaraldehied is 'n kruisbindingsagent wat met amino groepe reageer, terwyl EDC 'n koppelingsagent is wat karboksie groepe aktiveer. Hierdie agente word algemeen gebruik vir die binding van biomolekules aan soliede matrikse. Die kovalent gekoppelde fluidMAG-E537D ß-Galaktosidase partikels is gekarakteriseer met betrekking tot behoue ensimatiese aktiwiteit en vermoë om laktose te bind en sies te verwyder vanuit 'n oplossing wat laktose bevat deur 'n eksterne magneetveld op die ensiem-partikel kompleks in oplossing toe te pas, nadat die binding van laktose plaasgevind het. Elke komponent van hierdie funksioneel geïmmobiliseerde ensiemkomplekse is ondersoek en geoptimaliseer met die doel om by te dra tot die ontwikkeling van 'n nuwe tegniek wat bekostigbaar en eenvoudig is vir die verwydering van laktose vanuit 'n oplossing vir die uiteindelike gebruik in die produksie van laktose-vrye melk. Resultate het getoon dat die glutaraldehied metode van ß-Gal kruisbinding op fluidMAG-Amine verkies word aangesien dit 'n verhoogde draerkapasiteit van proteïene op die partikels moontlik maak. Die glutaraldehied gekoppelde proteïene beskik ook oor twee keer meer aktiwiteit as die EDC gekoppelde proteïene. Die glutaraldehied gekoppelde fluidMAG-E537D ß -Gal kon 34 % van die laktose teenwoordig in 'n 0.2 nmol/L laktose oplossing sies verwyder. Hierdie het dus die potensiële gebruik van hierdie nuwe metode in die voedselproduksie industrie bevestig.

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Les biopiles enzymatiques sont considérés comme des systèmes potentiellement utilisables pour la production d'énergie renouvelable dans des marchés niches. Une biopile est constituée de deux électrodes associées à des enzymes, catalyseurs biologiques, qui permettent la production d'énergie électrique à partir de réactions chimiques d'oxydoréduction. Ce travail présente la réalisation d'une biopile alcool/oxygène, au sein de laquelle l'alcool est oxydé à l'anode par l'alcool déshydrogénase alors que l'oxygène moléculaire est réduit en eau à la cathode par l'enzyme laccase, en présence de médiateurs spécifiques. L'objectif de ce travail a été tout d'abord de développer des bioélectrodes avec des enzymes immobilisées de manière à minimiser la quantité de biocatalyseur et augmenter sa stabilité. Dans un second temps, l'assemblage de biocathodes et de bioanodes a permis de fabriquer des biopiles à alcool macroscopique et microfluidique. Différentes poudres de carbone combinées à des polymères ont été utilisées pour immobiliser les enzymes et les médiateurs par encapsulation selon diverses configurations. Des analyses électrochimiques ont permis de mettre en évidence l'influence importante de certains paramètres comme la nature du carbone et du polymère, le pH et la température sur les performances des bioélectrodes. Une fois assemblées dans les configurations classique ou microfluidique, ces bioélectrodes ont conduit à des systèmes électrochimiques de génération d'énergie délivrant une densité de puissance maximale de 300μW/cm2 à 0,61V pour la biopile macroscopique et de 45μW/cm2 à 0,5V pour le système microfluidique. / Enzymatic biofuel cells (BFC) are systems of great interest for the production of renewable energy in niche markets. A BFC consists of two electrodes associated with enzymes as catalysts allowing energy production from oxydoreduction reactions. This work is devoted to the development of an alcohol/oxygen BFC for which alcohol is oxidized at the anode by alcohol dehydrogenase while molecular oxygen is reduced to water at the cathode by laccase, in the presence of specific mediators. The objective of this work was first to develop bioelectrodes with immobilized enzymes in order to minimize the amount of biocatalyst and increase its stability. In a second step, biocathodes and bioanodes were assembled to make macroscopic and microfluidic alcohol BFCs. Various carbon powders combined to polymers were used to immobilize enzymes and mediators in various configurations by entrapment. Electrochemical analysis have highlighted the significant influence of certain parameters like the nature of polymer and carbon, the pH or the temperature on the bioelectrodes performances. Once assembled in classical or microfluidic configurations, these bioelectrode led to electrochemical energy generation systems delivering a maximum power density of 300μW/cm2 at 0,61V for the macroscopic BFC and 45μW/cm2 at 0,5V for the microfluidic system.

L'un des avantages majeurs des réactions de bioconversion résulte du fait que le milieu réactionnel des biocatalyseurs est l'eau. Ce dit avantage constitue l'une des principales limitations de ces réactions de bioconversion lorsqu'il s'agit de substances lipophiles non solubles dans l'eau connue les stérols. L'efficacité d'un procédé de bioconversion de substances lipophiles solides dépend essentiellement du contact et de 1'interaction entre le biocatalyseur et ce substrat lipophile. Les solutions proposées dans la littérature font appel à des solvants et des produits chimiques de natures souvent toxiques, inflammables et explosives. Ces solutions décrites font perdre à la bioconversion son caractère de biotechnologie blanche. Dans ce travail, nous avons montré qu'en plus de ses avantages connus, l'immobilisation passive de biocatalyseurs au sein d'un support poreux peut favoriser l'interaction cellule-substrat lipophile et augmenter le taux de bioconversion sans utiliser de solvants et de produits chimiques. La réaction modèle étudiée est le clivage de la chaine latérale des stérols par des mycobactéries en vue de l'obtention des androsténones précurseurs naturels des stéroïdes, molécules à forte valeur biologique ajoutée. Le support d'immobilisation le plus performant a été le fruit sec de Luffa cylindrica. Par rapport aux supports organiques classiques tels que les gels de polyacrylamide, les mousses de polyuréthane, la silicone et les plastiques, le fruit sec de Luffa cylindrica présente les avantages suivants : (i) c'est un produit naturel, (ii) biodégradable, (iii) peu onéreux, (iv) non toxique pour les microorganismes, (v) stable du point de vue mécanique et thermique, (vi) et réutilisable. / One of the major advantages of using biocatalysts in organic synthesis is that water constitutes the reaction medium. However, water becomes a serious problem when bioconversion deals with lipophilic compounds, in particular those poorly soluble in water such as sterols. Bioconversion of lipophilic compounds depends on the close contact between the hydrophobic substrate and the biocatalyst. Increasing this contact requires usually the use of huge amounts of chemical which are often toxic, flammable and explosive. In this work, we showed that passive cell immobilization in porous materials can increase the contact between microorganisms and lipophilic substrates without using chemicals. The side chain cleavage of sterols was studied as a model multistep microbial bioconversion of lipophilic compounds. This reaction allows the production of androstenones which are the natural precursors of steroids. Among the studied immobilization carriers, the dried fruit of Luffa cylindrica was the most efficient Compared to other organic support carrier such as alginate beads, polyurethane foams, silicones and plastics, the dried fruit of Luffa cylindrica is advantageous since it is natural, renewable, biodegradable, cheap, mechanically strong, free of toxicity and it doesn't need a chemical pretreatment.

Immobilisation of functional entities, such as, enzymes, onto solid supports, as a means of facilitating their removal from the surrounding environment and subsequent regeneration has been in practice for many decades. This work focuses on the immobilisation and analysis of three-walled (pendant armed), cyclen based receptor complexes immobilised onto a silica surface for the purpose of sequestering aromatic anions from aqueous solution: Si-GPS-[Cd(Trac)](ClO4)2, Si-GPS-[Cd(DiPTrac)](ClO4)2, and Si-GPS-[Cd(TriPTrac)](ClO4)2 were the immobilised receptors used. Initially, synthesis of a three-walled model receptor, [Cd(TracHP12)](ClO4)2, that is not bound to silica yet mimics the properties of the silica anchored receptor complexes with a hydroxypropyl pendant arm was effected. Aromatic anion binding constant measurements were made on the model receptor using 1H NMR monitored titrations in DMSO-d6 which showed that, in comparison to the first generation four-walled receptors, the removal of one of the pendant arms did not affect the binding capability of the receptor's cavity significantly. It was shown that the binding strength correlated well with the pKa of the particular anion with, for example, p-hydroxybenzoate > m-hydroxybenzoate > o-hydroxybenzoate. The precursor to this receptor was then immobilised onto a silica surface and subjected to metal ion uptake studies to gauge its coordination properties with a number of divalent metal(II) ions: Cd(II), Pb(II), Zn(II), Cu(II) and Ca(II). The three Cd(II) coordinated receptor complexes mentioned above were then subjected to inclusion studies with a number of aromatic anions in aqueous conditions whereupon a reversal of the previously mentioned trend, i.e. o-hydroxybenzoate > m-hydroxybenzoate > p-hydroxybenzoate was observed. This indicated that the presence of water in the system changes the hydrogen bonding mode of the host-guest complexes, and was a major discovery arising from this work.